Smoking is a widely accepted practice in Iraqi and sometimes is
associated with socializing, sharing and male identity. A smoker defined
as a person who has smoked 10 or more cigarettes per day continually for
at least one year, while non-smoker is a person who has no previous
smoking experience (Shah, 2012)". Additionally, smokers have a
fivefold increased risk of cancer of the larynx and oral cavity, but one
third of all cancers of the kidneys and pancreas are attributing to
smoking as well. The risk of smoking having a heart attack is more than
twice that of nonsmokers and smokers risk for sudden cardiac death is
two to four times the risk of nonsmokers (Bashar, 2004)". Other
study found that water pipe tobacco smoking negatively affects lung
function and may be as harmful as cigarette smoking. In spite of these
deleterious health effects, water pipe smoking is widely believed to be
a less harmful form of tobacco smoking, and a safer alternative to
cigarette smoking (Akl, 2011)".

Hephaestin is predicted to be a trans membrane protein with a
molecular weight of approximately 130 kDa (Syed, 2002)". The
predicted amino acid sequence of human hephaestin is 50% identical and
68% similar to the sequence of human ceruloplasmin (Syed, 2002)".
Hephaestin is involved in the metabolism of iron. It is a trans membrane
copper-dependent peroxidase responsible for transporting dietary iron
from intestinal enterocytes into the circulatory system (Chen,
2006)". No previous study referred to level of hephaestin in
tobacco cigarette and water pipe smokers . Iron is an essential metal
for hemoglobin synthesis of erythrocytes, oxidation-reduction reactions,
and cellular proliferation, whereas excess iron accumulation causes
organ dysfunction through the production of reactive oxygen species
(ROS) (Kohgo, 2008)". The vast majority of body iron (at least 2.1
g in humans) is distributed in the hemoglobin of red blood cells and
developing erythroid cells and serves in oxygen transport. Significant
amounts of iron are also present in macrophages (up to 600 mg) and in
the myoglobin of muscles (~300 mg), whereas excess body iron (~1 g) is
stored in the liver (Olsson, 2008)". Transferrin is the major
plasma protein involved in transport of iron (Dales, 2008)".
Binding of iron by transferrin, results in striking conformational
changes in the two lobes (Lewis, 2002)". The delivery of
transferrin-bound iron to cells is dependent upon the expression of
transferrin-binding molecules, which are known as transferrin receptors
(Dales, 2008)". Thus, serum transferrin has considerable reserve
capacity to bind iron, called unsaturated iron binding capacity (UIBC)
(Fukuhara, 1996)". The aim of the present study is to evaluate the
effect of Tobacco Cigarette and water pipe smoking on hephaestin and
other biochemical and hematological parameters.

MATERIALS AND METHODS

Blood samples were collected from three groups of 30 cigarette
smokers, 30 waterpipe smokers and 30 non-smokers (control group) whose
ages ranged between (18-35) years. The study was done at College Health
and Medical Technical, Department of Medical analysis. The study was
performed from October 2013 to January 2014. The medical history was
taken. None of the participants had any chronic diseases.Venous blood
samples were drawn about 10 ml of venous blood was aspirated by
disposable syringe, two ml of this blood was transferred to EDTA
container tube, then mixed gently using blood shaker to prevent clotting
of the blood. This blood was used for estimation of complete blood count
(CBC), the rest of the blood was allowed to clot for 10-15 min. at room
temperature, centrifuged for (10) min. at (3000rpm). to separate serum
which was transferred into a new tube and frozen at(-20c[degrees]).

The Hb and other hematological parameters are measured by
Hematocrit. The serum hephaestin was measured by Enzyme Linked
Immunosorbent Assay(CUSABIO BIOTECH COM.).

Serum TIBC and iron level were measured by spectrophotometric
methods supplied by Human kits. To calculate the unsaturated iron
binding capacity (UIBC), the serum iron concentration was subtracted
from the TIBC.

UIBC = TIBC--Serum iron concentration

Transferrin can be estimated indirectly from the TIBC value by the
following equation (Mehde, 2013)".

Transferrin ([micro]g/dl) = 0.7 x TIBC ([micro]g/dl)

The percentage of saturation of transferrin with iron is determined
by the following equation:

% Saturation of transferrn with iron = Serum Iron/TIBC x 100

All statistical analysis in the study was performed using SPSS
version 19.0 for Windows (Statistical Package for Social Science, Inc.,
Chicago, IL, USA). Expressive analysis was presented the mean and
standard deviation of variables. The significance of differences between
mean values was assessed by Student t-test. The probability p < 0.05
was considered statistically significant, while p > 0.05 was referred
to statistically insignificant.

RESULT AND DISCUSSIONS

The Mean [+ or -] SD of age for water pipe smokers was [24.97 [+ or
-] 4.17] and for cigarette smokers was [28.03 [+ or -] 5.42] and for the
control group was [26.40 [+ or -] 4.47]. There were a non-significant
differences at p > 0.05 in water pipe and cigarette smokers when
compared with that found in control group, Table 1.

The WBC and LYM are showed a significant difference [p < 0.05]
in smokers groups when compared with that found in control groupas shown
in Table 2, table 3 showed a statistically increase in the WBC from a
mean value of [4.91 * [10.sup.3] cell/ml] in nonsmokers group to about
[5.99 * [10.sup.3] cell/ml], [5.93 * 103 cell/ml], for water pipe
smokers, cigarette smokersgroups respectively. There were a
non-significant differences [p > 0.05] in MID and GRA when compared
smokers groups with that found in control group Table 2.

Table 3 showed a statistically increase in the MID [%] from a mean
value of [9.31 [+ or -] 1.91%] in nonsmokers group to about [12.36 [+ or
-] 4.69%], [9.88 [+ or -] 1.54%], for water pipe smokers, cigarette
smokers groups respectively.

The present findings are conflicting to those of Baronchelli
(Baronchelh, 1952)" and Minuth (Minuth, 1957)". who reported
eosinopenia following cigarette smoking, on the other hand our result
are similar to the findings of Shenwai M, Aundhakar M (Shenwai,
2012)", and Tell G.S. & Grimm RH et al (Grimm, 1985)"who
described increased total leucocyte count in smokers who have started
smoking in recent times (Grimm, 1985)". Marked leucocytosis has
been reported by several other researchers even in subjects smokingless
than 10 cigarettes per day (Whitehead, 1995)".

As a result of the our study illustrates that there is a continuous
effect of smoking on total leucocyte count especially water pipe smokers
.The lymphocytosis may be due to chronic tissue damage and inflammation
produced by toxic smoke Products. This agrees with the findings of
Silvermann NA et al that leucocytosis in smokers is essentially
attributable to an increased lymphocyte count and as well of the
'T' lymphocytes (Silverman, 1974)". Chronic tissue damage
may be a probable mechanism for the increased leucocyte count in water
pipe smokers. Smoking (Tobacco Cigarette and waterpipe) have an
irritation effect on the respiratory with resultant chronic
inflammation. This probably explains the leukocytosis in smokers.

Table 4 ,5showed a statistically increase [p < 0.01] in the RBC
and HGBin smokers groups when compared with that found in control group.
The HCT[%] was significantly elevated [p < 0.05] in smokers groups
when compared with that found in control group. Mean of platelets for
smokers group was [267.18 * [10.sup.3] cell/ml] and for nonsmokers was
[291.53 * [10.sup.3] cell/ml]. There was significantly low [p < 0.05]
in smokers groups when compared with that found in control group. The
present study didn't find any significant difference in MCV, MCH,
MCHC, RDW and MPV between smokers group and nonsmokers group. The
current result agreement with Whithead TD et al, hemoglobin levels and
PCV increase significantly in smokers group more than 10 cigarettes per
day (Whitehead, 1995)"., while other study though the mean values
for all these parameters in smokers was found to be greater than
nonsmokers, but the difference is not statistically significant
(Shenwai, 2012)". Smoking is also considered as a major cause of
polycythemia (Attchison, 1998)".

Table 5 demonstrates a significant increase in RBC,HGB and HCT[%]
in water pipe smokers group (p < 0.01) when compared tonon-smokers
group. As our knowledge no previous study referred to these results.
Smoking is known cause of increase in hemoglobin (Hb) concentration,
that is believed to be mediated by exposure of carbon monoxide. Carbon
monoxide binds to Hb to form carboxyhemoglobin, an inactive form of
hemoglobin having no oxygen carrying capacity. Carboxyhemoglobin also
shifts the Hb dissociation curve in the left side, resulting in a
reduction in ability of Hb to deliver oxygen to the tissue. To
compensate the decreased oxygen delivering capacity, smokers maintain a
higher hemoglobin level than non-smokers (4h) (Arthur, 2006)".

Iron, TIBC, UIBC, transferrin ([micro]g/dl) showed a significant
increase (p < 0.01) and significant decrease in the mean of %
Saturation of transferrin with iron level showed in water pipe smokers
group when compared to cigarette smokers and non-smokers groups as shown
in table 6.Unusual iron status can be certainly clarified by the sign of
inflammation and the transfusions carried out. A possiblecause for this
portent could be improved intestinal iron absorption caused by water
pipe smoking--induced mucosal damage. Carbon monoxide has a higher
distribution coefficient paralleled to oxygen and enzyme in the human
body that generates carbon monoxide is hemeoxygenase which is found in
all cells and breaks down heme (Dunn, 2001)". While carbon monoxide
is not expressed it binds to hemoglobin, which is the principal
oxygen-carrying compound in blood. The traditional belief is that carbon
monoxide toxicity arises from the formation of carboxyhemoglobin, which
decreases the oxygen-carrying ability of the blood and inhibits the
transport, distribution, and use of oxygen by the body (Fan,
2009)".

Total iron-binding capacity (TIBC) is performed by drawing blood
and measuring the maximum amount of iron that it can carry, which
circuitously measures transferrin, since transferrin is the most dynamic
carrier. Total iron-binding capacity is a measure of the maximum
concentration of iron in serum bound to protein. Transferrin saturation
is nothing but present of saturation value with iron (Yamanishi,
2003)". Another mechanism includes effects on the mitochondrial
respiratory enzyme chain that is accountable for effective tissue
consumption of oxygen. Carbon monoxide binds to cytochrome oxidase with
less affinity than oxygen, so it is probable that it requires
significant intracellular hypoxia before binding (Kalantar-Zaden,
2003)". As our knowledge no previous study referred to these result
in water pipe smokers.

Hepcidin, a circulating peptide hormone is essential in systemic
iron regulation. It is produced by the liver is secreted into plasma,
and cleared by the kidneys. Hepcidin controls iron levels by interacting
directly with FPN1 in duodenal enterocytes, hepatocytes, and macrophages
resulting in internalization and degradation of FPN1 (Nemeth,
2005)". This blocks cellular iron export. When iron stores are
high, hepcidin is formed in the liver and transported to intestine which
effects in the blockage of iron delivery from the plasma via FPN1
degradation. Therefore, shedding of enterocytes results in iron loss.
While iron stores are low, Hepcidin production is inhibited, enabling
FPN1 to transmission iron from enterocytes to the plasma and therefore
dietary iron distribution in the body (Nemeth, 2005)".

Conclusions:

In summary, it can be concluded that water pipe smoking is
associated with increased WBC counts and increased serum hephaestin,
change of lymphocyte count and MID [%], TIBC and Transferrin contributes
to the increased risk of infection and neoplasia in smokers.
Thereforewater pipe smoking is considered as one of the major avoidable
risk factors for cardiovascular diseases and death. Also, the risk of
infection had increased in water pipe smoking more than cigarette
smokers smoking. This fact is of immense importance for the young
smokers who are else free from other predisposing factors like obesity,
hypertension, diabetes etc.